The EGFR contributes to the pathogenesis of many human cancers, including colorectal cancer (CRC), which is the third most commonly diagnosed cancer and the third leading cause of cancer deaths in the US for both men and women. The EGFR has become a major therapeutic target in many cancers. The EGFR neutralizing monoclonal antibody, cetuximab, is approved for the treatment of advanced CRCs that contain wild-type KRAS; however, only 15% of individuals with wild-type KRAS CRC respond to cetuximab, and individuals with mutant KRAS CRC do not respond to cetuximab. The challenge in cancer research I propose to address is: why has EGFR blockade in CRC (and other solid tumors) had such modest clinical benefit. I propose that the inability to more effectively block the EGFR in CRC is due, at least in part, to three issues: 1) an incomplete understanding of the complexity of EGFR signaling triggered by its seven mammalian ligands; 2) inadequate predictive preclinical models and 3) the emergence of drug resistance. By addressing each of these three issues, the overall goal of this revised application is to significantly advance the diagnosis, treatment and monitoring of individuals with CRC. Our focus is CRC, viewed from the perspective of membrane-proximal EGFR-related events. We anticipate that advances we make will be applicable to other solid tumors in which EGFR signaling plays a prominent role. Based on our recent finding that the Egfr inhibitor, Lrig1, marks a distinct population of colonic stem cells and acts as a tumor suppressor, along with the use of unique reporter mice (Lrig1-Apple, Egfr-EmGFP), we propose to link key events in colonic neoplasia to stem cells and Egfr- related events. Our lab has developed a robust model of colonic neoplasia: within 50 days of inducing loss of one Apc allele in Lrig1-expressing colonic stem cells, multiple, highly dysplastic colonic adenomas arise that can be monitored by colonoscopy and novel PET imaging probes. These mice will be treated with the first available mouse Egfr neutralizing antibody. Findings in mouse adenomas will be related to human adenomas. Using MulltiOmyx and DISSECT, we will examine the tumor landscape at single cell resolution and deconstruct tumor heterogeneity. Using a newly developed 3D culture system, we have discovered a novel mode of cetuximab resistance via increased WNT signaling due to marked upregulation of a long con-coding RNA not previously linked to CRC. We will further elucidate the mechanism of this resistance and advance these findings clinically. We will further examine a new mode of signaling by EGFR and its ligands via exosomes and test whether EGFR-containing exosomes act as a decoy to reduce the amount of EGFR antibody delivered to tumors. We will harness the tools and resources at Vanderbilt University, Vanderbilt-Ingram Cancer Center and Vanderbilt's GI Specialized Programs of Research Excellence (SPORE) to advance this work.